Associations between marine invertebrates and endosymbiotic bacteria are increasingly recognized as widespread and of biological importance (McKenzie and Kelly 1994). Examples of marine invertebrates utilizing symbiotic bacteria include sponges, annelids, bivalve molluscs, cephalopods, and echinoderms (Kelly et al. 1995). Of the five extant classes of echinoderms, all are known to harbor symbiotic bacteria directly below the cuticle, lying in or within the folds of the dermal lamallae (McKenzie and Kelly 1994; Kelly et al. 1995). Holland and Nealson (1978) first described these bacteria, known as subcuticular bacteria (SCB). Little is known of the biology and role of SCB relative to their hosts, but chemoautotrophic symbionts are found in sea-urchin guts, and some feather stars (criniods) are known to have bacteria enclosed in their pinnules (McKenzie and Kelly 1994).
Many morphological studies of SCB have been done. Transmission electron microscope (TEM) observations of SCB revealed that they are Gram-negative bacilli (McKenzie and Kelly 1994). The SCB found in Amphipholis (Microphiopholis) gracillima (Ophiuroidea) (hereinafter "A. gracillima") were described by McKenzie and Kelly (1994) as single straight rods, with membrane-bound vacuoles. All SCB examined by McKenzie and Kelly (1994) had simple, thin capsules. None of the bacteria observed in A. gracillima tissues exhibited evidence for flagella or pili, nor showed any type of mobility (McKenzie and Kelly 1994; Kelly and McKenzie 1995).
SCB could play a role in defense against bacterial infestation during tissue regeneration. Since brittlestars exhibit the ability to autotomize and then regenerate body parts, brittlestars are candidates for pathogenic microbial colonization (Bryan et al. 1994). Bryan et al. (1994) proposed that antimicrobial compounds, produced by echinoderms, would help prevent infection by pathogenic bacteria and thus prevent surface fouling of epithelial tissue that can deplete respiratory capacity, impede elimination of waste products, and reduce tissue elasticity. Lubchenco et al. (1991) suggested SCB and echinoderms may have evolved a mutualistic relationship that provides for protection against colonization of other bacterial species.
McKenzie and Kelly (1994) showed that regenerating tissue, such as damaged ophiuroid arm tips, are quickly colonized by SCB. When the arms of Amphipholis gracillima were surgically removed, SCB colonized the wound closure within 1 hour and almost completely covered the regenerating stump within 24 hours (Dobson 1988). Due to this rapid proliferation, SCB may limit colonization of pathogenic bacteria due to competitive exclusion, production of a compound which exhibits antimicrobial activity, or both.
Despite the aforementioned study of SCB in the prior art, the isolation of putative SCB from intact and regenerating brittlestars and subsequent characterization of those bacteria for ability to chemically inhibit the growth of pathogenic bacteria in culture has not been described. Isolation and characterization of putative SCB are highly desirable given the need to determine whether such bacteria produce antimicrobial compounds and given the broad utility of any such compounds produced by the bacteria as potential antimicrobials.